Plasmid deoxyribose nucleic acid (DNA) is a circular or looped format of genetic material located within all living cells. Purified plasmid DNA is widely studied and exploited in molecular biology as a tool to determine gene structure and function. One use of the purified plasmid DNA is the mapping of the human genome. The purified plasmid DNA must be sufficiently pure so as to permit additional investigational manipulations involving restriction enzymes and sequencing.
Two procedures to isolate plasmid DNA are alkaline lysis (Maniatis et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)) and boiling (Akela et al, Rapid Isolation and Sequencing of Double Stranded Plasmid DNA, Biotechniques 14: 726-728 (1993) and Holmes et al., A Rapid Boiling Method for the Preparation of Bacterial Plasmids, Anal. Biochem. 114: 193-197 (1981)). Many of these methods use hazardous chemicals such as phenol/chloroform, guanidine hydrochloride and the like. Detergents used in these procedures often contain a phenyl ring that interferes with ultraviolet (UV) spectrophotometric analysis of proteins or nucleic acids. Some detergents, such as sodium dodecylsulfate (SDS), leave a residue that inhibits cutting of the plasmid DNA with restriction enzymes.
Reduction or elimination of phenol in the preparation of isolated plasmid DNA is highly desirable. Wang et al, Simplified Large-Scale Alkaline Lysis Preparation of Plasmid DNA With Minimal Use of Phenol, Biotechniques, Vol. 17, No. 1, pp 27-28 (1994).
Holmes et at. discloses a boiling method for plasmid isolation that uses Triton X-100. Triton X-100 has the following shortcomings:
(a) Due to a phenyl ring in its structure, Triton X-100 interferes with direct, spectral estimations of protein and nucleic acid concentration. PA1 (b) Triton X-100 is not a definable, pure compound. Rather, it is a mixture containing various chain lengths of ethoxylated isooctylphenol. The use of undefined mixtures is not compatible with Good Manufacturing Practice (GMP) procedures often required for processing of pharmaceuticals. PA1 (c) Polyethoxylated nonionic detergents such as Triton X-100 frequently are contaminated or become contaminated with peroxides upon aging. This contaminant can be especially destructive to the biological integrity and activity of proteins and nucleic acids. PA1 (d) In order to maintain solutions of Triton X-100 above the cloud point of 30.degree. C., external heat must be continuously applied which adds mechanical complexity and further increases the risk of damaging certain sensitive proteins and nucleic acids.
During isolation of plasmid DNA from bacteria, a large number of double stranded DNA templates are routinely generated. Some of the DNA templates contain significant amounts of secondary structures that often terminate the sequencing reactions prematurely making it difficult to read and analyze the DNA. One way to overcome this problem is to sub-clone the DNA fragment into a single stranded vector such as M13. Alternately, high temperature sequencing can be performed using, e.g., Taq polymerase. However, high temperature sequencing has been shown to be prone to error. See, for example, Lee-Jackson et al., Artifactual Frame Shift p53 Mutation at Codon 249 Detected with Cyclyst.TM. DNA Sequencing Method, Biotechniques 15: 363-4 (1993).
Many detergents such as SDS that include an ester undergo alkaline hydrolysis that makes them unstable in alkaline solutions such as those produced by mixing SDS and sodium hydroxide. Due to this lack of stability, the sodium hydroxide and SDS solution should be made fresh before every use which renders alkaline SDS solutions useless for inclusion in a kit to isolate purified plasmid DNA. See Wang et al.
A method of isolating purified plasmid DNA, solution and kit that overcome at least some of the above-identified shortcomings is highly desirable.